Therapy for hepatitis C virus (HCV) infection has advanced rapidly with the recent approval of several direct-acting antivirals. However, most of the DAAs in clinical use or clinical trials target the same stage of HCV replication cycle and are associated with rapid emergence of drug-resistant viral mutations. In addition, different HCV genotypes and clinical conditions may also require adjustment of treatment regimen. Therefore, there is still an ongoing need to develop new HCV inhibitors that target different stages of the HCV replication cycle, such as entry and assembly. We developed a quantitative high-throughput screen (qHTS) assay platform with a cell-based HCV infection system. The highly sensitive assay can be miniaturized to 1536-well format for screening of large-scale chemical libraries. we performed a large-scale quantitative high-throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of about 350,000 chemicals and a library of approved pharmaceutical collection of about 3,000 compounds (the NPC library) for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial 3,000 molecule showing inhibitory activity for further structural analyses and functional assays. We were able to assign the majority of these compounds to specific stage(s) in the HCV replication cycle. These small molecules represent a diversity of chemotypes that are potential drug-like lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. From the NPC library, we identified chlorcyclizine HCl (CCZ), an over-the-counter drug for allergy symptoms, and related compounds as potent inhibitors of HCV infection. In an effort to optimize the CCZ class of compounds, we started a chemical/structural modification campaign, centered around chlorcyclazine, which resulted in optimized, non-chiral, nontoxic CCZ analogues with improved anti-HCV potency and pharmacokinetics that provide good coverage in liver at very reasonable doses. Lead compounds inhibited HCVsc infection without affecting HCVpp entry or HCV replication in the replicon assay, which is similar to that of CCZ, suggesting unaltered mechanism of action. Lead compounds showing overall improved properties, will be selected for further preclinical development efforts with the aim of moving compounds of this series toward anti-HCV human clinical trials. In the above HTS of the MLSMR library, we also identified an aryloxazole-based anti-HCV hit. Structure-activity relationship studies revealed several compounds exhibiting EC50 values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis. With the increasing pipeline of anti-HCV drugs in development, there is a need to establish an in vitro system to assess the efficacy of various combination therapies. To achieve this aim, we established an infectious full-length HCV system expressing a luciferase reporter (HCVcc-Luc) for combination testing of synergy, additivity or antagonism. We tested combinations between protease, NS5A, and nucleotide NS5B inhibitor classes in both Con1b replicon and HCVcc-Luc systems. Combinations between different classes show consistency across the two viral assay platforms and different computational softwares, MacSynergyII and CalcuSyn. Combinations between NS5A and nucleotide NS5B inhibitors were synergistic, while combinations of protease inhibitors with the other two classes were additive to antagonistic. Theoretically additive combinations were indeed additive in the HCVcc-Luc system. Subsequent application to combinations between these DAA classes and host-targeting agent cyclosporin A demonstrated additive to synergistic effects. Combinations between an entry inhibitor (S)-chlorcyclizine with these DAA classes or cyclosporin A were highlyly synergistic. Together, our results demonstrate applicability of this infectious HCV system in combination testing. This system allows for in vitro investigation of novel combinatorial regimens, including antivirals targeting the entry or assembly stage of the HCV life cycle. Invitrogens RevolutionTM technology (by Life Technologies) that can efficiently inhibit DNA mismatch repair when introduced into cells, has been applied to CHO DG44 cells hereby generating a genetically diverse cell population. Such a technology has been successful in generating cell lines produce high levels of antibodies. We applied this approach to express the hepatitis C virus envelope glycoproteins E1 and E2 (subunit vaccine) at a high level as potential recombinant vaccine production We have utilized the RevolutionTM-treated CHO K1 cell to express hepatitis C virus proteins (E1 and E2) and have screened for high producing cell lines for potential subunit vaccine production. We have isolated three clones with an E1/E2 construct and two with a truncated E2 construct that show high protein productivity and quality. These clones have been shown to produce and secrete the E2 glycoprotein that can be detected in the culture supernatant by various monoclonal antibodies against E2. In some of the clones, ELISA can also detect E1 glycoprotein in the supernatant. Additionally, these recombinant viral proteins have also been tagged with a hexahistidine tag, which allows us to purify the recombinant proteins from the cell supernatant. After further analysis of the purified recombinant proteins from each clone, we selected three of the five clones for progressive expansion and adaptation for large-scale production. Currently, we are completing the progressive expansion and have successfully adapted the cell clones to serum-free adherent media and serum-free suspension culture. After a scaling up the culture, we plan to examine the protein productivity of the cell clones in controlled bioreactors and purify large quantity recombinant protein to evaluate the immune response of this subunit vaccine in various animal models and evaluate its suitability for further vaccine development.